Exploration of the Radiobiology of a Novel Targeted Alpha Therapy
Abstract
Purpose
A novel targeted alpha-particle therapy (TAT), 225Ac-MTI-201, targets the melanocortin 1 receptor (MC1R) which is highly expressed in metastatic uveal melanoma (MUM) and is currently in a first-in-human clinical trial. The favorable qualities of TAT include the large linear energy transfer (LET) and stochastic energy deposition leading to macromolecular damage and production of reactive oxygen species (ROS) through oxidative stress in addition to direct DNA damage. A dichloro-dihydro fluorescein diacetate (DCFH-DA) assay was performed to measure ROS production in MEL270 MUM cells that endogenously express MC1R with and without TAT via the relative fluorescence unit (RFU). Another experiment was performed without cells, using 2′,7′ dichlorofluorescein (DCF), with and without TAT, to quantify radio-bleaching damage to the dye. RFU vs time graphs were generated for MEL270 cells with correction factors for radio-bleaching and radiation-related signal detected. A third experiment quantified ATP production which is associated with biological ROS.
Methods
The DCFH-DA assays were performed on MEL270 cells that were preincubated with DCFH-DA for 45 mins, followed by a 30s incubation with 1.5 µCi TAT, 3X washes, and incubated with indicator-free medium over a time-course with fluorescence measurements. Cell-free experiments were conducted using 1.5 µCi for 30 s, and 0.045 µCi ATP production was measured using the same treatment regimen via Cell-TiterGlo.
Results
The dye + TAT experiment determined 13% reduction in RFU, indicating damage to dye, and a 3% increase in RFU was detected during the incubation with 0.045 µCi activity. These changes were used to correct the cellular data to quantify biological ROS relative to radioactivity generated ROS. A decrease in biological ROS relative to untreated controls was observed following treatment, correlating with decreased ATP production.
Conclusion
As mitochondrial ATP production generates biological ROS, the decreased RFU signal may be explained from damage done to the mitochondria by 225Ac-MTI-201.